Cryptococcus neoformans is a major pathogen in immunocompetant as well as immunocompromised patients including those with AIDS in both the developed as well as the developing world. Our long-term objective is to test the hypothesis that molecular regulators of the virulence factor laccase affects the virulence of Cryptococcus neoformans. The specific hypothesis behind the present proposal is that a virulence associated DEAD-box protein, Vad1, identified by insertional mutagenesis, is an important regulator of laccase and virulence in C. neoformans. This is based on the following observations. First, deletion of VAD1 results in loss of virulence and accelerated clearance of C. neoformans from lung in mouse models. Second, differential display has shown that deletion of VAD1 results in altered transcription of a number of genes in addition to laccase. Finally, deletion of one of the genes showing VAD1-dependent transcription, PCK1, exhibited attenuated virulence in a mouse model in spite of retained laccase activity. In the last project period, we completed a detailed biochemical analysis of the mechanism of Vad1-dependent degradation of the MFalpha mating pheromone and the role of this regulation in mating. We also continued our studies of the role of autophagy in cryptococcal pathogenesis by detailing the role of ATG8 regulation of this important virulence-related phenotype. In addition, we identified a role for copper acquisition in cryptococcal pathogenesis and determined a role of increased copper quota in cryptococcal metabolism. Finally, we showed that the immune response was attenuated to vad1 delta mutants using a mouse clearance model. In conclusion, our studies in the most recent period have shown new methods of virulence regulation in the AIDS-related pathogen, Cryptococcus neoformans that could have applications toward novel drug and control strategies for this important pathogen.